Ix 81 fluoview fv1000 confocal laser scanning system

The labeling of surface GABA_A receptors and confocal immunofluorescence microscopy analysis were performed according to published procedure [14 (link)]. Briefly, cells on coverslips were fixed for 3 min with 4% formaldehyde on ice and incubated in 100 μL of HEPES buffer (HEPES 25 mM, NaCl 140 mM, KCl 5.4 mM, CaCl₂ 1.8 mM, glucose 15 mM, pH = 7.4) containing mouse monoclonal anti-α1 antibody (Millipore #MAB339) and rabbit monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Abcam #ab76020), or rabbit polyclonal anti-γ2 antibody (Synaptic Systems #224,003) and mouse monoclonal anti-Na⁺/K⁺-ATPase, a plasma membrane marker (Santa Cruz Biotechnology #sc-48345) for 1.5 h. The primary antibodies were used at 1:300 dilutions. Cells were then incubated with 100 μL of HEPES buffer containing an Alexa 488-conjugated secondary antibody (1:600) and an Alexa 594-conjugated secondary antibody (1:600). Afterwards, cells were permeabilized with saponin (0.2%) for 10 min and incubated with DAPI (1 µg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60X objective collected images using FV10-ASW software. Quantification of the fluorescence intensity after background correction was done using the ImageJ software from the NIH.

To label cell surface GABA_A receptors, primary neurons that were cultured on coverslips, were fixed with 2% paraformaldehyde in DPBS, blocked with goat serum for 0.5 h at room temperature, and labeled with 100 μL of appropriate anti-α1 (Synaptic Systems, catalog #: 224203), β2/3 (Millipore, catalog #: 05-474), or γ2 (Synaptic Systems, catalog #: 224003) antibodies (1:200) for 1 h without detergent permeabilization. Afterwards, they were incubated at room temperature with 500 μL (1:400) of Alexa 594-conjugated goat anti-rabbit antibody (ThermoFisher, catalog #: A11037), or Alexa 594-conjugated goat anti-mouse antibody (ThermoFisher, catalog #: A11032) for 1 h. Afterwards, cells were permeabilized with saponin (0.2%) for 5 min and incubated with DAPI (1 μg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60× objective was used to collect images using FV10-ASW software. Quantification of the fluorescence intensity was achieved using the ImageJ software from the NIH.